Fig 1: Macrophages engulf apoptotic cells (AC) carrying viable SARS-CoV-2.(A) Immunoblot of caspase-8 and caspase-3 cleavage in Calu-3 and Vero CCL81 cells in response to infection with SARS-CoV-2 for 48 hr (CoV2, detected with rabbit anti-Spike), UV irradiation (UV), or freezing and thawing (F/T). (B) Flow cytometric analysis of caspase-3 cleavage (Cl-caspase-3) in Calu-3 cells unstimulated (Cntrl) or infected with CoV2 for 48 hr. The pan-caspase inhibitor z-VAD-FMK (zVAD; 20 µM) or vehicle (DMSO) was added to cell cultures after viral adsorption. Representative histogram, geometric mean fluorescence intensity (MFI), and frequency of Cl-caspase-3+ cells (gated on total cells/single cells) are shown. The black line on the histogram represents the control sample labeled with secondary antibody only. (C) Flow cytometric analysis of apoptosis in Vero CCL81 cells in response to UV light irradiation or 48 hr post-infection with CoV2, assessed by annexin V (FITC) and viability dye (Zombie NIR) co-staining. Representative histogram and frequency of each population (pre-gated on total cells/single cells) are shown. (D) Cytotoxicity analysis by measurement of lactate dehydrogenase (LDH) release on the supernatants of Vero CCL81 cells in response to UV light irradiation or 48 hr post-infection with CoV2. (E, F) Representative histological findings in post-mortem lung tissue from COVID-19 patients obtained by ultrasound-guided minimally invasive autopsy. (E) Immunofluorescence images of tissue samples labeled with anti-Cl-caspase-3 (red), anti-dsRNA (green), and stained with Hoechst (nuclei, blue) for the detection of caspase-3 activation in infected epithelia (COVID-19, bottom panels) compared to samples from control patients (Cntrl, upper panels). Tissues were scanned by wide-field epifluorescence imaging. Scale bar: 20 µm, asterisks: erythrocytes. The MFI of Cl-caspase-3 of at least 300 cells is shown (n=3 individuals per group). Technical control for secondary antibodies background is shown in Figure 1—figure supplement 1. (F) Representative immunofluorescence image of a COVID-19 patient tissue sample labeled for the detection of efferocytosis in situ and superimposed with differential interference contrast. Samples were labeled with anti-CD68 (detecting macrophages, green), anti-cytokeratin18 (CK-18, detecting epithelial cells, white), anti-Spike (detecting SARS-CoV-2, red), and stained with Hoechst (nuclei, blue). Bottom panels show higher magnification of ( a,b) CD68+ macrophage with CK-18 and Spike labeling in the cytosol and (c) CD68+ only macrophage. White arrows: CK-18+ cells; asterisks: erythrocytes. Tissues were scanned by wide-field epifluorescence imaging. Scale bar: 20 µm; scale bar for insets: 10 µm. (G) Quantification by TCID50 of SARS-CoV-2 viral loads in Vero CCL81 cells infected for 48 hr. Viral loads were estimated for pre-sorted (total cells) or annexin V+ (Ann+ cells) cells isolated by magnetic separation. (H) Uptake of apoptotic Vero CCL81 cells (labeled with CTFR) in response to UV irradiation (UV-AC) or infection with SARS-CoV-2 for 48 hr (Cov2-AC) by human monocyte-derived macrophages (labeled with CellTrace Violet (CTV)). Macrophages were co-incubated with AC for 2 hr, and internalization was assessed by flow cytometry. Representative plot and percentage of engulfment (gated on total cells/single cells/live cells/CTV+/ CTV+CTFR+ cells) are shown. Boxes represent the mean of five (B, D), four (C, H), or three (G) biological replicates, and error bars are ± S.E.M. Each biological replicate is shown as a circle. Significance was calculated by ANOVA (B–D), Mann-Whitney test (E), or Student’s t-test (G, H). *p<0.05, **p<0.001, ***p<0.0001, comparing the indicated groups. Data shown are from one representative out of two (A, C, G) or three (B, D, H) experiments performed independently with similar results. Figure 1—source data 1.Source data for Figure 1.